This invention relates to piperidino-phenyl amino squarate and thiadiazole dioxide xcex23 adrenergic receptor agonists useful for the treatment of metabolic disorders related to insulin resistance or hyperglycemia (typically associated with obesity or glucose intolerance), atherosclerosis, gastrointestinal disorders, neurogenic inflammation, glaucoma, ocular hypertension, and frequent urination; and are particularly useful in the treatment or inhibition of type II diabetes.
The subdivision of xcex2 adrenergic receptors (xcex2-AR) into xcex21- and xcex22-AR has led to the development of xcex21- and xcex22- antagonists and/or agonists which have been used in the treatment of cardiovascular disease and asthma. The recent discovery of xe2x80x9catypicalxe2x80x9d receptors, later called xcex23-AR, has led to the development of xcex23-AR agonists that are potentially useful as antiobesity and antidiabetic agents. For recent reviews on xcex23-AR agonists, see: 1. Strosberg, A. D., Annu. Rev. Pharmacol. Toxicol, 1997, 37, 421; 2. Weber, A. E., Ann. Rep. Med. Chem., 1998, 33, 193; 3. Kordik, C. P. and Reitz, A. B., J. Med. Chem., 1999, 42, 181; 4. Weyer, C., Gautier, J. F., and Danforth, E., Diabetes and Metabolism, 1999, 25, 11.
Compounds that are potent and selective xcex23 agonists, may be potentially useful antiobesity agents. Low levels or lack of xcex21 and xcex22-agonistic properties will minimize or eliminate the adverse side effects that are associated with xcex21 and xcex22 agonistic activities, i.e. increased heart rate, and muscle tremor, respectively. Early developments in the xcex23-agonist field are described in European patent 427480, U.S. Pat. Nos. 4,396,627, 4,478,849, 4,999,377, 5,153,210. Although the early developments purport to claim compounds with greater xcex23-AR selectivity over the xcex21- and xcex22-AR. However, clinical trials in humans with those early developed xcex23-agonists have, so far, not been successful.
More recently, potent and selective human xcex23 agonists have been described in several patents and published applications: WO 98/32753, WO 97/46556, WO 97/37646, WO 97/15549, WO 97/25311, WO 96/16938, and WO 95/29159; European Patents 659737, 801060, 714883, 764640, and 827746; and U.S. Pat. Nos. 5,561,142, 5,705,515, 5,436,257, and 5,578,620. These compounds were evaluated in a Chinese hamster ovary (CHO) cell model, an assay that predicts the effects expected in humans. These assays utilize cloned human xcex23 receptors, expressed in CHO cells (see refs. Granneman, et al., Mol. Pharmacol., 1992, 42, 964; Emorine, et al., Science, 1989, 245,1118; Liggett, 50 Mol. Pharmacol., 1992, 42, 634).
xcex23-AR agonists also are useful in controlling urinary incontinence. It has been shown that relaxation of the bladder detrusor is under beta adrenergic control (Li, J. H., Yasay, G. D. and Kau, S. T., xe2x80x9cBeta-adrenoceptor subtypes in the detrusor of guinea-pig urinary bladderxe2x80x9d, Pharmacology, 1992, 44, 13-18). Several laboratories have provided recent experimental evidence that activation of the xcex23 receptor subtype by norepinephrine is responsible for relaxation of the urinary bladder in a number of animal species, including humans (Yamazaki Y., et al., xe2x80x9cSpecies differences in the distribution of the xcex2-AR subtypes in bladder smooth musclexe2x80x9d, Br. J. Pharmacol.,1998, 124, 593-599).
Urge urinary incontinence is characterized by abnormal spontaneous bladder contractions that can be unrelated to bladder urine volume. Urge urinary incontinence is often referred to as hyperactive or unstable bladder. Several etiologies exist and fall into two major categories, myogenic and neurogenic. The myogenic bladder is usually associated with detrusor hypertrophy secondary to bladder outlet obstruction, or with chronic urinary tract infection. The neurogenic bladder is associated with an uninhibited micturition reflex, in which an upper motor neuron disease is usually the underlying cause. In either case, the disease is characterized by abnormal spontaneous contractions that result in an unusual sense of urinary urgency and involuntary urine loss. At present, the most common therapy for hyperactive bladder involves the use of antimuscarinic agents to block the action of the excitatory neurotransmitter acetylcholine. While effective in neurogenic bladders, their utility in myogenic bladders is questionable. In addition, due to severe dry mouth side-effects associated with antimuscarinic therapy, the patient compliance with these agents is only approximately 30 percent.
In the bladder, xcex23-AR agonists activate adenylyl cyclase and generate cAMP through the G-protein coupled xcex23-AR. The resulting phosphorylation of phospholamban/calcium ATPase enhances uptake of calcium into the sarcoplasmic reticulum, thereby decreasing intracellular calcium resulting in an inhibition of bladder smooth muscle contractility.
It is suggested therefore, that activation of the xcex23-AR in the urinary bladder will inhibit abnormal spontaneous bladder contractions and be useful for the treatment of bladder hyperactivity. Note that unlike the antimuscarinics, xcex23-AR agonists would be expected to be active against both neurogenic and myogenic etiologies.
Despite these recent developments there is still no single therapy available for the treatment of type II diabetes (NIDDM), obesity, atherosclerosis, gastrointestinal disorders, neurogenic inflammation, frequent urination and related diseases. A potent and selective xcex23-AR agonist is therefore highly desirable for the potential treatment of these disease states.
This invention provides compounds of Formula I having the structure 
(a) phenyl optionally substituted with 1-3 Y groups;
(b) a 5-6 membered heterocyclic ring having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups;
(c) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups; or
(d) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, having a second phenyl ring fused to the heterocyclic ring, optionally substituted with 1-2 Y groups; 
Y is hydroxy, halogen, cyano, xe2x80x94SOmR2, xe2x80x94SOnNR2R3, xe2x80x94NHSO2R2, xe2x80x94NR2R3, alkyl of 1-10 carbon atoms, cycloalkyl of 3-8 carbon atoms, alkoxy of 1-10 carbon atoms, arylalkoxy, xe2x80x94COR2, or xe2x80x94CO2R2;
X is xe2x80x94OCH2xe2x80x94 or a bond;
R1 is
(a) alkyl of 1-10 carbon atoms, optionally substituted with 1-5 groups selected from the group consisting of halogen; hydroxy; phenyl optionally mono- or di-substituted with Z; oxo; xe2x80x94CO2H; xe2x80x94CO2R2; amino; xe2x80x94NR2R3; and xe2x80x94NHCOR2;
(b) cycloalkyl of 3-8 carbon atoms;
(c) arylalkyl wherein the alkyl moiety contains 1-10 carbon atoms;
(d) heterocycle or heterocyclealkyl, wherein the alkyl moiety contains 1-6 carbon atoms, and the heterocycle is
i) a 5-6 membered heterocyclic ring having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups;
ii) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups; or
iii) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, having a second phenyl ring fused to the heterocyclic ring, optionally substituted with 1-2 Y groups;
R2 and R3 are each, independently, hydrogen, alkyl of 1-10 carbon atoms, or cycloalkyl of 3-8 carbon atoms;
Z is hydroxy, halogen, alkyl of 1-10 carbon atoms, xe2x80x94CO2R2, benzyloxy, xe2x80x94NHC(O)NHR2, xe2x80x94NR2R3, xe2x80x94OR2, xe2x80x94COR2, xe2x80x94S(O)mR2; or xe2x80x94S(O)nNR2R3;
m=0-2;
n=1-2
or a pharmaceutically acceptable salt thereof, which are selective agonists at human xcex23 adrenergic receptors and are useful in treating or inhibiting metabolic disorders related to insulin resistance or hyperglycemia (typically associated with obesity or glucose intolerance), atherosclerosis, gastrointestinal disorders, neurogenic inflammation, glaucoma, ocular hypertension, and frequent urination; and are particularly useful in the treatment or inhibition of type II diabetes.
Pharmaceutically acceptable salts can be formed from organic and inorganic acids, for example, acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic, camphorsulfonic, and similarly known acceptable aids when a compound of this invention contains a basic moiety. Salts may also be formed from organic and inorganic bases, such as alkali metal salts (for example, sodium, lithium, or potassium) alkaline earth metal salts, ammonium salts, alkylammonium salts containing 1-6 carbon atoms or dialkylammonium salts containing 1-6 carbon atoms in each alkyl group, and trialkylammonium salts containing 1-6 carbon atoms in each alkyl group, when a compound of this invention contains an acidic moiety.
The compounds of the instant invention all contain at least one asymmetric center. Additional asymmetric centers may be present on the molecule depending upon the nature of the various substituents on the molecule. Each such asymmetric center will produce two optical isomers and all such optical isomers, as separated, pure or partially purified optical isomers or racemic mixtures thereof, are included within the scope of the instant invention. Any enantiomer of a compound of the general Formula I may be obtained by stereospecific synthesis using optically pure starting materials of know configuration.
Alkyl and alkenyl include both straight chain as well as branched moieties. Halogen means bromine, chlorine, fluorine, and iodine. Aryl includes monocyclic or bicyclic aromatic carbocyclic groups such as phenyl and naphthyl. Benzyl is the preferred arylalkyl moiety.
As used herein, a heterocyclic ring is a ring confining 1-4 heteroatoms selected from N, O, and S, indicates a heterocycle which may be saturated, unstaurated, or partially unsaturated. The heterocyclic ring may be attached within structural Formula I by any carbon atom or appropriate heteroatom. It is understood that the heterocyclic ring does not contain heteroatoms in arrangements which would make them inherently unstable. For example, the term heterocyclic ring does not include ring systems containing Oxe2x80x94O bonds in the ring backbone. Preferred heterocyclic radicals include pyridinyl, thiophenyl, furanyl, benzothiophenyl, benzofuranyl, benzodioxolyl, quinolinyl, thiadiazolyl, thiazolyl, oxadiazolyl, carbazolyl, pyrrolyl, imidazolyl, benzimidazolyl, benzotriazolyl, 1,2,3,4-tetrahydroquinolyl, 1,2,3,4-tetrahydroisoquinolyl, and pyrazolyl.
One set of preferred compounds of this invention are the compounds of formula I wherein 
(a) phenyl optionally substituted with 1-3 Y groups;
(b) a 5-6 membered heterocyclic ring having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups;
(c) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups; or
(d) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, having a second phenyl ring fused to the heterocyclic ring, optionally substituted with 1-2 Y groups; 
Y is hydroxy, halogen, cyano, xe2x80x94SOmR2, xe2x80x94SOnNR2R3, xe2x80x94NHSO2R2, xe2x80x94NR2R3, alkyl of 1-10 carbon atoms, cycloalkyl of 3-8 carbon atoms, alkoxy of 1-10 carbon atoms, arylalkoxy, xe2x80x94COR2, or xe2x80x94CO2R2;
X is xe2x80x94OCH2xe2x80x94 or a bond;
R1 is
(a) alkyl of 1-10 carbon atoms, optionally substituted with 1-5 groups selected from the group consisting of halogen; hydroxy; phenyl optionally mono- or di-substituted with Z; oxo; xe2x80x94CO2H; xe2x80x94CO2R2; amino; xe2x80x94NR2R3; and xe2x80x94NHCOR2;
(b) cycloalkyl of 3-8 carbon atoms;
(c) arylalkyl wherein the alkyl moiety contains 1-10 carbon atoms;
(d) heterocycle or heterocyclealkyl, wherein the alkyl moiety contains 1-6 carbon atoms, and the heterocycle is
i) a 5-6 membered heterocyclic ring having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups;
ii) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups; or
iii) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, having a second phenyl ring fused to the heterocyclic ring, optionally substituted with 1-2 Y groups;
R2 and R3 are each, independently, hydrogen, alkyl of 1-10 carbon atoms, or cycloalkyl of 3-8 carbon atoms;
Z is hydroxy, halogen, alkyl of 1-10 carbon atoms, xe2x80x94CO2R2, benzyloxy, xe2x80x94NHC(O)NHR2, xe2x80x94NR2R3, xe2x80x94OR2, xe2x80x94COR2, xe2x80x94S(O)mR2; or xe2x80x94S(O)nNR2R3;
m=0-2;
n=1-2
or a pharmaceutically acceptable salt thereof.
A second set of preferred compounds of this invention are the compounds of formula I, wherein 
(a) phenyl optionally substituted with 1-3 Y groups;
(b) a 5-6 membered heterocyclic ring having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups;
(c) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups; or
(d) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, having a second phenyl ring fused to the heterocyclic ring, optionally substituted with 1-2 Y groups; 
Y is hydroxy, halogen, cyano, xe2x80x94SOmR2, xe2x80x94SOnNR2R3, xe2x80x94NHSO2R2, xe2x80x94NR2R3, alkyl of 1-10 carbon atoms, cycloalkyl of 3-8 carbon atoms, alkoxy of 1-10 carbon atoms, arylalkoxy, xe2x80x94COR2, or xe2x80x94CO2R2;
X is xe2x80x94OCH2xe2x80x94 or a bond;
R1 is
(a) alkyl of 1-10 carbon atoms, optionally substituted with 1-5 groups selected from the group consisting of halogen; hydroxy; phenyl optionally mono- or di-substituted with Z; oxo; xe2x80x94CO2H; xe2x80x94CO2R2; amino; xe2x80x94NR2R3; and xe2x80x94NHCOR2;
(b) cycloalkyl of 3-8 carbon atoms;
(c) arylalkyl wherein the alkyl moiety contains 1-10 carbon atoms;
(d) heterocycle or heterocyclealkyl, wherein the alkyl moiety contains 1-6 carbon atoms, and the heterocycle is
i) a 5-6 membered heterocyclic ring having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups;
ii) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, optionally substituted with 1-2 Y groups; or
iii) a phenyl fused heterocycle having 1-4 heteroatoms selected from O, N, and S, having a second phenyl ring fused to the heterocyclic ring, optionally substituted with 1-2 Y groups;
R2 and R3 are each, independently, hydrogen, alkyl of 1-10 carbon atoms, or cycloalkyl of 3-8 carbon atoms;
Z is hydroxy, halogen, alkyl of 1-10 carbon atoms, xe2x80x94CO2R2, benzyloxy, xe2x80x94NHC(O)NHR2, xe2x80x94NR2R3, xe2x80x94OR2, xe2x80x94COR2, xe2x80x94S(O)mR2; or xe2x80x94S(O)nNR2R3;
m=0-2;
n=1-2
or a pharmaceutically acceptable salt thereof.
More preferred compounds of this invention are those compounds of formula I, wherein 
(a) phenyl optionally substituted with 1-3 Y groups; 
Y is hydroxy, halogen, cyano, xe2x80x94SOmR2, xe2x80x94SOnNR2R3, xe2x80x94NHSO2R2, xe2x80x94NR2R3, alkyl of 1-10 carbon atoms, cycloalkyl of 3-8 carbon atoms, alkoxy of 1-10 carbon atoms, arylalkoxy, xe2x80x94COR2, or xe2x80x94CO2R2;
X is xe2x80x94OCH2xe2x80x94;
R1 is
(a) alkyl of 1-10 carbon atoms, optionally substituted with 1-5 groups selected from the group consisting of halogen; hydroxy; phenyl optionally mono- or di-substituted with Z; oxo; xe2x80x94CO2H; xe2x80x94CO2R2; amino; xe2x80x94NR2R3; and xe2x80x94NHCOR2;
R2 and R3 are each, independently, hydrogen or alkyl of 1-10 carbon atoms;
Z is hydroxy, halogen, alkyl of 1-10 carbon atoms, xe2x80x94CO2R2, benzyloxy, xe2x80x94NHC(O)NHR2, xe2x80x94NR2R3, xe2x80x94OR2, xe2x80x94COR2, xe2x80x94S(O)mR2; or xe2x80x94S(O)nNR2R3;
m=0-2;
n=1-2
or a pharmaceutically acceptable salt thereof.
Specifically preferred compounds of this invention are:
a) 4-((2S)-2-hydroxy-3-{1-[4-(4-octylamino-1,1-dioxo-1H-1.lambda(6).-[1,2,5]thiadiazol-3-ylamino)-phenyl]-piperidin-4-ylamino}-propoxy)-phenol;
b) 4-{(2S)-3-[1-(4-{4-[2-(4-fluoro-phenyl)-ethylamino]-1,1-dioxo-1H-1.lambda(6).-[1,2,5]thiadiazol-3-ylamino}-phenyl)-piperidin-4-ylamino]-2-hydroxy-propoxy)-phenol;
c) (S)-4-{2-hydroxy-3-[1-(4-{4-[2-(4-methoxy-phenyl)-ethylamino]-1,1-dioxo-1H-1.lambda(6).-[1,2,5]thiadiazol-3-ylamino)-phenyl)-piperidin-4-ylamino]-propoxy}-phenol;
d) methyl (2R)-2-{[2-(4-{4-[((2R)-2-hydroxy-2-{4-hydroxy-3-[(methylsulfonyl)amino]phenyl}ethyl)amino]piperidin-1-yl}anilino)-3,4-dioxocyclobut-1-en-1-yl]amino}-3-phenylpropanoate;
e) methyl 2-{[4-(4-{4-[((2R)-2-hydroxy-2-{4-hydroxy-3-[(methylsulfonyl)amino]phenyl}ethyl)amino]piperidin-1-yl}anilino)-1,1-dioxido-1,2,5-thiadiazol -3-yl]amino}-3-phenylpropanoate;
f) methyl {[4-(4-(4-[((2R)-2-hydroxy-2-{4-hydroxy-3-[(methylsulfonyl)amino]phenyl}ethyl)amino]piperidin-1-yl}anilino)-1,1-dioxido-1,2,5-thiadiazol -3-yl]amino}acetate;
or a pharmaceutically acceptable salt thereof.
The compounds of this invention were be prepared according to the following schemes from commercially available starting materials or starting materials which can be prepared using literature procedures. These schemes show the preparation of representative compounds of this invention.
In Scheme 1 the 4-nitrophenyl-4xe2x80x2-piperidone 1 is known in the literature (Synthesis 1981, 606-608) or readily prepared by methods commonly known to those skilled in the art. Reductive amination with ammonium acetate and sodium cyanoborohydride yields the amine which is protected with di-tert-butyl dicarbonate and a base to give the Boc-protected (Boc=tert-butoxy carbonyl) amine 2. Hydrogenation of the nitro group with a metal catalyst in a suitable solvent yields the aniline which is coupled with 3,4-diethoxy-1,1-dioxo-1,2,5-thiadiazole in an ethanol or a suitable solvent to give 3 (J. Org. Chem. 1975, 40, 2743). Substitution of the ethoxy group with a suitably 
functionalized amine (RNH2) in refluxing alcohol results in 4. Deprotection of the amine with trifluoroacetic acid in a chlorinated hydrocarbon, then coupling of the amine with an epoxide in a solvent like dimethyl formamide or methanol at 40 to 60 degrees Celsius results in the product 5. Deprotection of the phenol in Scheme 1 with tetrabutyl ammonium fluoride in a suitable solvent yields the product of formula I.
The synthesis of squarate analogs is described in Scheme 2. Starting with the known protected piperidone 6 (Synthesis 1981, 606-608) the nitro group is hydrogenated with a metal catalyst to give the aniline. Reaction with the commercially available 3,4-diethoxy-3-cyclobutene -1,2-dione in a suitable solvent yields 7. Substitution of the ethoxy 
group with a suitably functionalized amine (RNH2) in refluxing solvent like ethanol or methanol results in 8. Deprotection of the ketone with acid yields 9. Reductive amination of the ketone with amine 10 sodium triacetoxyborohydride in DMF/acetic acid yields the product of formula I.
Thiadiazole dioxide compounds similar to the compounds in Scheme 2 where the thiadiazole dioxide replaces the squarate, are synthesized in a similar manner. Starting 
with nitro compound 6, hydrogenation to the aniline then coupling with 3,4-diethoxy-1,1-dioxo-1,2,5-thiadiazole in a suitable solvent yields 11. Substitution of the ethoxy group with a suitably functionalized amine (RNH2) in refluxing solvent like ethanol or methanol results in 12. Deprotection of the ketone with acid yields 13. Reductive amination of the ketone with amine 10 sodium triacetoxyborohydride in DMF/acetic acid yields the product of formula I.
The compounds of this invention are useful in treating metabolic disorders related to insulin resistance or hyperglycemia, typically associated with obesity or glucose intolerance. The compounds of this invention are therefore, particularly useful in the treatment or inhibition of type II diabetes. The compounds of this invention are also useful in modulating glucose levels in disorders such as type I diabetes.
The ability of the compounds of this invention to treat or inhibit disorders related to insulin resistance or hyperglycemia was confirmed with representative compounds of this invention in the following standard pharmacological test procedures, which measured the binding selectivity of the xcex21-, xcex22-, and xcex23-AR. Binding to the receptors was measured in Chinese Hamster ovary (CHO) cells that were transfected with xcex21-, xcex22-, and xcex23-AR""s. The following briefly summarizes the procedures used and results obtained.
Transfection of CHO cells with xcex21- and xcex22-AR: CHO cells were transfected with human xcex21- or xcex22-AR as described in Tate, K. M., Eur. J. Biochem., 1991, 196, 357-361.
Cloning of Human xcex23-AR Genomic DNA: cDNA was constructed by ligating four polymerase chain reaction (PCR) products using the following primers: an ATG-Narl fragment, sense primer 5xe2x80x2-CTTCCCTACCGCCCCACGCGCGATC3xe2x80x2 and anti-sense primer 5xe2x80x2CTGGCGCCCAACGGCCAGTGGCCAGTC3xe2x80x2; a Narl-Accl fragment, 5xe2x80x2TTGGCGCTGATGGCCACTGGCCGTTTG3xe2x80x2 as sense and 5xe2x80x2GCGCGTAGACGAAGAGCATCACGAG3xe2x80x2 as anti-sense primer; an Accli-Styl fragment, sense primer 5xe2x80x2CTCGTGATGCTCTTCGTCTCACGCGC3xe2x80x2 and anti-sense primer 5xe2x80x2GTGAAGGTGCCCATGATGAGACCCAAGG3xe2x80x2 and a Styl-TAG fragment, with sense primer 5xe2x80x2CCCTGTGCACCTTGGGTCTCATCATGG3xe2x80x2 and anti-sense primer 5xe2x80x2CCTCTGCCCCGGTTACCTACCC3xe2x80x2. The corresponding primer sequences are described in Mantzoros, C. S., et.al., Diabetes, 1996, 45, 909-914. The four fragments are ligated into a pUC 18 plasmid (Gibco-BRL) and sequenced. Full-length xcex23-AR clones (402 amino acids) containing the last 6 amino acids of hxcex23-AR are prepared with the xcex23-xcex2ARpcDNA3 from ATTC.
Binding Procedure: Clones expressing receptor levels of 70 to 110 fmoles/mg protein were used in the test procedures. CHO cells were grown in 24-well tissue culture plates in Dulbecco""s Modified Eagle Media with 10% fetal bovine serum, MEM non-essential amino acids, Penicillin-Streptomycin and Geneticin. On the day of test procedure, growth medium was replaced with preincubation media (Dulbecco""s Modified Eagle Media) and incubated for 30 minutes at 37xc2x0 C. Pre-incubation medium was replaced with 0.2 ml treatment medium containing DMEM media containing 250 xcexcM IBMX (isobutyl-1-methylxantine) plus 1 mM ascorbic acid with test compound dissolved in DMSO. Test compounds were assayed over a concentration range of 10xe2x88x929 M to 10xe2x88x925 M for xcex23-AR transfected cells and 10xe2x88x928 to 10xe2x88x924 M for xcex21-AR and xcex22-AR transfected cells. Isoproterenol (10xe2x88x925 M) was used as an internal standard for comparison of activity. Cells were incubated at 37xc2x0 C. on a rocker for 30 min with the xcex23-AR transfected cells and 15 min with xcex21-AR and xcex22-AR transfected cells. Incubation was stopped by the addition of 0.2N HCl and the acid was neutralized with 2.5N NaOH. The plates, containing the cells and neutralized media, were stored at xe2x88x9220 xc2x0 C. until ready to test for cAMP using the SPA test kit (Amersham).
Data Analysis and Results: Data collected from the SPA test procedure were analyzed as percent of the maximal isoproterenol response at 10xe2x88x925 M. Activity curves were plotted using the SAS statistical and graphics software. EC50 values were generated for each compound and the maximal response (IA) exhibited by each compound was compared to the maximal response of isoproternol at 10xe2x88x925 M from the following formula:
IA=% activity compound % activity isoproterenol
Shown in Table I are the xcex23-AR EC50 and IA values for the representative compounds of this invention that were evaluated in this standard pharmacological test procedure. Compounds of the present invention were active at the xcex23-AR as shown by these results. The compounds of this invention were considerably less active, if at all, at the xcex21- and/or xcex22-AR.
Based on these results, representative compounds of this invention have been shown to be selective xcex23-AR agonists. They are therefore useful in treating metabolic disorders related to insulin resistance or hyperglycemia (typically associated with obesity or glucose intolerance), atherosclerosis, gastrointestinal disorders, neurogenic inflammation, glaucoma, ocular hypertension, and frequent urination; and are particularly useful in the treatment or inhibition of type II diabetes, and in modulating glucose levels in disorders such as type I diabetes. As used herein, the term modulating means maintaining glucose levels within clinically normal ranges.
As used in accordance with this invention, the term providing an effective amount means either directly administering such a compound of this invention, or administering a prodrug, derivative, or analog which will form an effective amount of the compound of this invention within the body.
It is understood that the effective dosage of the active compounds of this invention may vary depending upon the particular compound utilized, the mode of administration, and severity of the condition being treated, as well as the various physical factors related to the individual being treated. As used in accordance with this invention, satisfactory results may be obtained when the compounds herein are administered at a daily dosage of 0.1 mg to 1 mg per kilogram of body weight, preferably in divided doses two to six times per day, or in a sustained release form. For most large mammals, the total daily dosage is from 3.5 mg to 140 mg. It is preferred that the administration of one or more of the compounds herein begin at a low dose and be increased until the desired effects are achieved.
Such doses may be administered in any manner useful in directing the active compounds herein to the recipient""s bloodstream, including orally, via implants, parenterally (including intravenous, intraperitoneal and subcutaneous injections), rectally, intranasally, vaginally, and transdermally. For the purposes of this disclosure, transdermal administrations are understood to include all administrations across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administrations may be carried out using the present compounds, or pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).
Oral formulations containing the active compounds of this invention may comprise any conventionally used oral forms, including tablets, capsules, buccal forms, troches, lozenges and oral liquids, suspensions or solutions. Capsules may contain mixtures of the active compound(s) with inert fillers and/or diluents such as the pharmaceutically acceptable starches (e.g. corn, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc. Useful tablet formulations may be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidinone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starches and powdered sugar. Oral formulations herein may utilize standard delay or time release formulations to alter the absorption of the active compound(s).
In some cases it may be desirable to administer the compounds directly to the airways in the form of an aerosol.
The compounds of this invention may also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for administration by syringe include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The form must be sufficiently fluid to permit administration by syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository""s melting point, and glycerin. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.
The compounds of the present invention also possess utility for increasing lean meat deposition and/or improving lean meat to fat ratio in edible animals, i.e. ungulate animals and poultry.
Animal feed compositions effective for increasing lean meat deposition and for improving lean meat to fat ratio in poultry, swine, sheep, goats, and cattle are generally prepared by mixing the compounds herein with a sufficient amount of animal feed to provide from 1 to 1000 ppm of the compound in the feed. Animal feed supplements can be prepared by admixing 75% to 95% by weight of a compound of this invention with 5% to 25% by weight of a suitable carrier or diluent. Carriers suitable for use to make up the feed supplement compositions include the following: alfalfa meal, soybean meal, cottonseed oil meal, linseed oil meal, sodium chloride, cornmeal, cane molasses, urea, bone meal, corncob meal and the like. The carrier promotes a uniform distribution of the active ingredients in the finished feed into which the supplement is blended. It thus performs an important function by ensuring proper distribution of the active ingredient throughout the feed. When the supplement is used as a top dressing for the feed, the carrier likewise helps to ensure a uniform distribution of the active compound across the top of the dressed feed.
The preferred medicated swine, cattle, sheep and goat feed generally contain from 0.01 to 400 grams of active ingredient per ton of feed, the optimum amount for these animals usually being 50 to 300 grams per ton of feed. The preferred poultry and domestic pet feed usually contain 0.01 to 400 grams and preferably 10 to 400 grams of active ingredient per ton of feed.
For parenteral administration, the compounds described herein may be prepared in the form of a paste or a pellet and administered as an implant, usually under the skin of the head or ear of the animal in which an increase in lean meat deposition and/or an improvement in lean meat to fat ratio is sought. Parenteral administration involves injection of a sufficient amount of the compounds of the present invention to provide the animal with 0.001 to 100 mg/kg/day of body weight of the active ingredient. The preferred dosage for swine, cattle, sheep and goats is in the range of 0.001 to 50 mg/kg/day of body weight of active ingredient. The preferred dosage for poultry and domestic pets is usually in the range of 0.001 to 35 mg/kg/day of body weight.
Paste formulations can be prepared by dispersing the active compounds in pharmaceutically acceptable oils such as peanut oil, sesame oil, corn oil or the like. Pellets containing an effective amount of the compounds herein can be prepared by admixing these compounds with a diluent such as carbowax, carnuba wax, and the like, and a lubricant, such as magnesium or calcium stearate, can be added to improve the pelletizing process. It is recognized that more than one pellet may be administered to an animal to achieve the necessary dosage that will provide the desired increase in lean meat deposition and/or improvement in lean meat to fat ratio. Moreover, it has been found that implants may also be employed periodically during the animal treatment period in order to maintain the proper drug level in the animal""s body. For poultry and swine farmers, the method of this invention results in leaner animals.
The compounds of this invention are also useful in elevating the lean mass to fat ratio in domestic pets. For the pet owner or veterinarian who wishes to increase leanness and trim unwanted fat from pets, the present invention provides the means by which this can be accomplished.
The preparation of representative examples of this invention is described below.